Optimization of a LanthaScreen Kinase assay for NTRK1 (TRKA)

Optimization of a LanthaScreen Kinase assay for NTRK1 (TRKA) Overview This protocol describes how to develop a LanthaScreen kinase assay designed to detect and characterize kinase inhibitors. The development is performed in three steps: 1. Optimization of kinase concentration required to determine ATP K m,app. The assay is first performed at a high concentration of ATP (1 mm) against a dilution series of kinase in order to determine the amount of kinase required to elicit an approximately 80% change between the minimum and maximum TR-FRET emission ratios (the EC 80 value). 2. Determination of ATP K m,app. Using the concentration of enzyme determined in step 1, the assay is then performed against a dilution series of ATP in order to determine the amount of ATP required to elicit a 50% change between the minimum and maximum TR-FRET emission ratios (the EC 50 value). This concentration of ATP is referred to as the apparent K m value for ATP, or the ATP K m,app. 3. Optimization of kinase concentration required for assay at ATP K m,app. Using the ATP K m,app concentration of ATP determined in step 2, the kinase titration is repeated in order to determine the concentration of kinase required to elicit an approximately 80% change between the minimum and maximum TR-FRET emission ratios at the ATP K m,app concentration of ATP (the EC 80 value). This is the concentration of kinase that will be used in an assay to determine an IC 50 value for an inhibitor. Using the ATP and kinase concentrations determined above, the reaction is then performed in the presence of a dilution series of inhibitor, and the amount of inhibitor required to elicit a 50% change in TR-FRET ratio (the IC 50 ) is determined. The experiments described in this document can be performed over two days, with steps one and two being performed on the first day, and step three and the inhibitor IC 50 determination(s) being performed on the second day. The optimization presented here is designed to maximize sensitivity of the assay towards both ATP-competitive as well as non-atp competitive inhibitors. If desired, the assay can be performed at higher concentrations of ATP in order to be less sensitive towards ATP-competitive compounds. If such an assay is desired, step 1 is the only step that needs to be performed (at the chosen concentration of ATP) prior to performing the assay in the presence of inhibitors. The data presented in this document is example data that was generated at Invitrogen. Specific results may vary based upon a variety of factors including the specific activity of the kinase, or the particular plate reader being used. In particular, the Emission Ratio measured can vary greatly between instruments. However, the quality of the data generated should be comparable to the data presented here. If you are reproducing the work presented in this document you should move between the various steps using the values determined in your experiments. If you are having trouble reproducing the data presented here, please do not hesitate to contact Invitrogen Technical Services or your Invitrogen representative. MAN0013677 1

Materials Required Description Part Name Catalog # Notes Kinase Reaction Buffer 5X Kinase Buffer PV3189 (4 ml of 5X) (1) Additives: None n.a. Kinase NTRK1 PV3144 (10 µg) Antibody LanthaScreen Tb-PY20 PV3552 (25 µg) PV3553 (1 mg) Substrate Fluorescein-Poly GT PV3610 (1 ml) (3) Antibody Dilution Buffer TR-FRET Dilution Buffer PV3574 (100 ml) (4) 500 mm EDTA Kinase Quench Buffer P2825 (1 ml) 10 mm ATP 10 mm ATP PV3227 (500 µl) (1) The kinase reaction buffer is supplied as a 5x concentrated stock. Prepare a 1x solution from this stock as described below. The 1x kinase reaction buffer is stable at room temperature. (2) The PY20 antibody is supplied at an approximate concentration of either 0.5 or 1 mg/ml. The molecular weight of the antibody is 150 kd. Thus, the stock concentration of 0.5 mg/ml antibody is 3.35 µm, or 3350 nm, and 1 mg/ml antibody is 6.7 µm, or 6700 nm. (3) The substrate is supplied at a concentration of 30 µm. (4) The antibody dilution buffer does not contain EDTA. EDTA is added separately, prior to addition of antibody. (2) Preparing the 1x Kinase Reaction Buffer Prepare a 1x solution of kinase reaction buffer from the 5x Kinase Buffer stock (listed above) by adding 4 ml of 5x stock to 16 ml H 2 O to make 20 ml of 1x kinase reaction buffer. General Assay Conditions Kinase reactions are performed in a 10 µl volume in low-volume 384-well plates. Typically, Corning model 3676 (black) or 3673 (white) plates are used. The concentration of substrate in the assay is 200 nm, and the 1x kinase reaction buffer consists of 50 mm HEPES ph 7.5, 0.01% BRIJ-35, 10 mm MgCl 2, and 1 mm EGTA, plus any additional additives that may be required for a specific kinase. Kinase reactions are allowed to proceed for 1 hour at room temperature before a 10 µl preparation of EDTA (20 mm) and Tb-labeled antibody (4 nm) in TR-FRET dilution buffer are added. The final concentration of antibody in the assay well is 2 nm, and the final concentration of EDTA is 10 mm. The plate is allowed to incubate at room temperature for at least 30 minutes before being read on a plate reader configured for LanthaScreen TR-FRET. Plate Readers The data presented in this document were generated using a BMG Pherastar plate reader using the LanthaScreen filter block available from BMG. The assay can be performed on a variety of plate readers including those from Tecan (Ultra, Safire 2, and InfiniTE F500), Molecular Devices (Analyst and M5), and Perkin Elmer (EnVision, Victor, and ViewLux). Visit www.invitrogen.com/lanthascreen or contact Invitrogen Discovery Sciences technical support at 800-955-6288 (select option 3 and enter 40266), or email tech_support@invitrogen.com for more information on performing LanthaScreen assays on your particular instrument. Example Protocols The following example protocols describe the various steps using 16-point dilutions of the variable reagent (kinase, ATP, or inhibitor) in triplicate. 2

Step 1: Titration of Kinase at 1 mm ATP (1.1) In an appropriate tube or vial, prepare 2000 µl of kinase in 1x kinase reaction buffer at 2 times the highest concentration of kinase to be tested. In this example, 0.1 µg/ml (100 ng/ml) was the highest concentration of kinase to be tested, and the stock concentration of kinase was 370 µg/ml. Kinase: Stock = 370 µg/ml 1x = 0.1 µg/ml 2x = 0.2 µg/ml Add 1.1 µl of 370 µg/ml kinase to 1998.9 µl kinase reaction buffer. Keep the diluted kinase on ice until needed. (1.2) In a low-volume 384-well plate, fill each well in columns 1 3, rows 2 16 (B through P) with 5 µl kinase reaction buffer. Place 10 µl of the kinase solution prepared in step 1.1 in the top well of each column, and then perform a 2-fold serial dilution down the plate by removing 5 µl of kinase from the top well, adding this to the well below, mixing, and repeating with the next well below. Discard 5 µl of solution from the bottom well such that each well contains 5 µl of kinase solution. (1.3) In an appropriate container, prepare 1 ml of substrate and ATP in kinase reaction buffer at 2 times the final concentration of each that is desired in the assay. If a 1 ml solution is prepared in a plastic reagent reservoir (trough), then the next addition step can be performed using a multichannel pipette. Substrate: Stock = 30 µm 1x = 0.2 µm 2x = 0.4 µm ATP: Stock = 10 mm 1x = 1 mm 2x = 2 mm Kinase: 1.1 µl * 370 µg/ml = 2000 µl * 0.2 µg/ml 1998.9 µl kinase reaction buffer Substrate: 13.3 µl * 30 µm = 1000 µl * 0.4 µm ATP: 200 µl * 10 mm = 1000 µl * 2 mm 786.7 µl kinase reaction buffer Add 13.3 µl of 30 µm substrate and 200 µl of 10 mm ATP to 786.7 µl kinase reaction buffer. (1.4) Start the kinase reaction by adding 5 µl of the substrate + ATP solution prepared in step 1.3 to each well of the assay plate. (1.5) Cover the assay plate and allow the reaction to proceed for 1 hour at room temperature. (1.6) Prior to completion of the kinase reaction, prepare 1 ml of a solution of EDTA and Tb-labeled antibody at 2 times the desired final concentrations of each reagent in TR-FRET dilution buffer. The antibody is stable in EDTA for several hours, but because it is sensitive to high concentrations of EDTA, we recommend first adding the concentrated EDTA to the dilution buffer, mixing the solution well, and then adding the antibody before mixing further. 3

EDTA: Stock = 500 mm 1x = 10 mm 2x = 20 mm Antibody: Stock = 6700 nm 1x = 2 nm 2x = 4 nm EDTA: 40 µl * 500 mm = 1000 µl * 20 mm Antibody: 0.6 µl * 6700 nm = 1000 µl * 4 nm 959.4 µl TR-FRET Dilution Buffer Add 40 µl of 500 mm EDTA and 0.6 µl of 6700 nm antibody to 959.4 µl TR-FRET Dilution Buffer. Note:These calculations are made assuming that the stock concentration of antibody is 1 mg/ml. If the stock concentration is 0.5 mg/ml, then twice the indicated volume of antibody (1.2 µl) should be used to make the solution. (1.7) Add 10 µl of the Tb-antibody + EDTA solution prepared in step 1.6 to each well of the assay plate and mix briefly, either by pipette or on a plate shaker. (1.8) Cover the assay plate and incubate for 30 minutes at room temperature before reading on an appropriate plate reader. (1.9) Plot the resulting TR-FRET emission ratio against the concentration of kinase, and fit the data to a sigmoidal dose-response curve with a variable slope. Calculate the EC 80 concentration from the curve. The following equation can be used with GraphPad Prism software: F=80 logec50=logecf-(1/hillslope)*log(f/(100-f)) Y=Bottom+(Top-Bottom)/(1+10^((LogEC50-X)*HillSlope)) Alternatively, the amount of kinase needed to elicit an 80% change in TR-FRET response may be estimated from a visual inspection of the curve. It is important that the reactions in the next step of this protocol be performed at or below the EC 80 concentration of kinase. Figure 1: Example of Kinase Titration at 1 mm ATP Kinase Titration at 1 mm ATP Emission ratio 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0.001 0.01 0.1 1 10 100 [NTRK1] ng/ml The EC 80 value determined from the example data was 1.53 ng/ml kinase. Based on this result, 1.5 ng/ml kinase was used for the following step of this protocol. Note: Above 12.5 ng/ml kinase, a decrease was seen in the emission ratio due to the antibody associating with phosphotyrosines on the kinase itself rather than on phosphorylated product. These data points are not included on the graph above and were not used when calculating the EC 80 value for the kinase. 4

Step 2: Titration of ATP at the Initial EC 80 Concentration of Kinase to determine ATP K m,app. (2.1) In a small tube or vial, prepare 50 µl of a 2 mm ATP solution by adding 10 µl of 10 mm ATP to 40 µl of kinase reaction buffer. (2.2) In a low-volume 384-well plate, fill each well in columns 1 3, rows 2 through 16 (B through P) with 5 µl kinase reaction buffer. Place 10 µl of the 2 mm ATP solution prepared in step 2.1 in the top well of each column, and then perform a 2-fold serial dilution down the plate by removing 5 µl of ATP from the top well, adding this to the well below, mixing, and repeating with the next well below. Discard 5 µl of solution from the bottom well such that each well contains 5 µl of ATP solution in kinase reaction buffer. (2.3) In an appropriate container, prepare 1 ml of a solution of substrate and kinase in kinase reaction buffer at 2 times the final concentration of each that is desired in the assay. If a 1000 µl solution is prepared in a plastic reagent reservoir (trough), then the next addition step can be performed using a multichannel pipette. Most kinases are stable enough such that you may use the diluted stock of kinase prepared in step 1.1 to prepare this solution, as long as it has been kept on ice since being prepared earlier in the day. Substrate: Stock = 30 µm 1x = 0.2 µm 2x = 0.4 µm Kinase: Initial conc. = 200 ng/ml* 1x = 1.5 ng/ml 2x = 3 ng/ml * prepared in step 1.1 Add 13.3 µl of 30 µm substrate and 15 µl of 200 ng/ml kinase to 971.7 µl kinase reaction buffer. (2.4) Start the kinase reaction by adding 5 µl of the substrate + kinase solution prepared in step 2.3 to each well of the assay plate. (2.5) Cover the assay plate and allow the reaction to proceed for 1 hour at room temperature. (2.6) Prior to completion of the kinase reaction, prepare 1 ml of a solution of EDTA and Tb-labeled antibody at 2 times the desired final concentrations of each reagent in TR-FRET dilution buffer. The antibody is stable in EDTA for several hours, but because it is sensitive to high concentrations of EDTA, we recommend first adding the concentrated EDTA to the dilution buffer, mixing the solution well, and then adding the antibody before mixing further. EDTA: Stock = 500 mm 1x = 10 mm 2x = 20 mm Antibody: Stock = 6700 nm 1x = 2 nm 2x = 4 nm Substrate: 13.3 µl * 30 µm = 1000 µl * 0.4 µm Kinase: 15 µl * 200 ng/ml = 1000 µl * 3 ng/ml 971.7 µl kinase reaction buffer EDTA: 40 µl * 500 mm = 1000 µl * 20 mm Antibody: 0.6 µl * 6700 nm = 1000 µl * 4 nm 959.4 µl TR-FRET Dilution Buffer 5

Add 40 µl of 500 mm EDTA and 0.6 µl of 6700 nm antibody to 959.4 µl TR-FRET Dilution Buffer. Note:These calculations are made assuming that the stock concentration of antibody is 1 mg/ml. If the stock concentration is 0.5 mg/ml, then twice the indicated volume of antibody (1.2 µl) should be used to make the solution. (2.7) Add 10 µl of the Tb-antibody + EDTA solution prepared in step 2.6 to each well of the assay plate and mix briefly, either by pipette or on a plate shaker. (2.8) Cover the assay plate and incubate for 30 minutes at room temperature before reading on an appropriate plate reader. (2.9) Plot the resulting TR-FRET emission ratio against the concentration of ATP, and fit the data to a sigmoidal dose-response curve with a variable slope. Calculate the EC 50 concentration from the curve. This is the ATP K m,app for your kinase under these assay conditions. Figure 2: Example of ATP K m,app Determination Curve 0.3 ATP Km,app Determination FRET ratio 0.2 0.1 0.0 0.01 0.1 1 10 100 1000 10000 [ATP] um The EC 50 value determined from the example data was 222.8 µm ATP. Based on this result, 223 µm ATP was used for the following step of this protocol. 6

Step 3: Titration of Kinase at ATP K m,app. (3.1) In an appropriate tube or vial, prepare 2000 µl of kinase in kinase reaction buffer at 2 times the highest concentration of kinase to be tested. In this example, 0.1 µg/ml (100 ng/ml) was the desired highest concentration of kinase to be tested, and the stock concentration of kinase was 370 µg/ml. Calculation: Kinase: Stock = 370 µg/ml 1x = 0.1 µg/ml 2x = 0.2 µg/ml Add 1.1 µl of 370 µg/ml kinase to 1998.9 µl kinase reaction buffer. Keep the diluted kinase on ice until needed. (3.2) In a low-volume 384-well plate, fill each well in columns 1 3, rows 2 through 16 (B through P) with 5 µl of kinase reaction buffer. Place 10 µl of the kinase solution as prepared above in the top well of each column, and then perform a 2-fold serial dilution down the plate by removing 5 µl of kinase from the top well, adding this to the well below, mixing, and repeating with the next well below. Discard 5 µl of solution from the bottom well such that each well contains 5 µl of kinase solution. (3.3) In an appropriate container, prepare 1 ml of a solution of substrate and ATP in kinase reaction buffer at 2 times the final concentration of each reagent desired in the assay. If a 1000 µl solution is prepared in a plastic reagent reservoir (trough), then the next addition step can be performed with a multichannel pipette. Substrate: Stock = 30 µm 1x = 0.2 µm 2x = 0.4 µm ATP: Stock = 10 mm 1x = 223 µm 2x = 446 µm Kinase: 1.1 µl * 370 µg/ml = 2000 µl * 0.2 µg/ml 1998.9 µl kinase reaction buffer Substrate: 13.3 µl * 30 µm = 1000 µl * 0.4 µm ATP: 44.6 µl * 10 mm = 1000 µl * 446 µm 942.1 µl kinase reaction buffer Add 13.3 µl of 30 µm substrate and 44.6 µl of 10 mm ATP to 942.1 µl kinase reaction buffer. (3.4) Start the kinase reaction by adding 5 µl of the substrate + ATP solution prepared in step 3.3 to each well of the assay plate. (3.5) Cover the assay plate and allow reaction to proceed for 1 hour at room temperature. (3.6) Prior to completion of the kinase reaction, prepare 1 ml of a solution of EDTA and Tb-labeled antibody at 2 times the desired final concentrations of each reagent in TR-FRET dilution buffer. The antibody is stable in EDTA for several hours, but because it is sensitive to high concentrations of EDTA we recommend first adding the concentrated EDTA to the dilution buffer, mixing the solution well, and then adding the antibody before mixing further. 7

EDTA: Stock = 500 mm 1x = 10 mm 2x = 20 mm Antibody: Stock = 6700 nm 1x = 2 nm 2x = 4 nm EDTA: 40 µl * 500 mm = 1000 µl * 20 mm Antibody: 0.6 µl * 6700 nm = 1000 µl * 4 nm 959.4 µl TR-FRET Dilution Buffer Add 40 µl of 500 mm EDTA and 0.6 µl of 6700 nm antibody to 959.4 µl TR-FRET Dilution Buffer. Note:These calculations are made assuming that the stock concentration of antibody is 1 mg/ml. If the stock concentration is 0.5 mg/ml, then twice the indicated volume of antibody (1.2 µl) should be used to make the solution. (3.7) Add 10µL of the Tb-antibody + EDTA solution prepared in step 3.6 to each well of the assay plate and mix briefly, either by pipette or on a plate shaker. (3.8) Cover the assay plate and incubate for 30 minutes at room temperature before reading on an appropriate plate reader. (3.9) Plot the resulting TR-FRET emission ratio against the concentration of ATP, and fit the data to a sigmoidal dose-response curve with a variable slope. Calculate the EC 80 concentration from the curve. The following equation can be used with GraphPad Prism software: F=80 logec50=logecf-(1/hillslope)*log(f/(100-f)) Y=Bottom+(Top-Bottom)/(1+10^((LogEC50-X)*HillSlope)) Alternatively, the amount of kinase needed to elicit an 80% change in TR-FRET response may be estimated from a visual inspection of the curve. It is important that the reactions performed to determine the IC 50 value of an inhibitor be performed at or below the EC 80 concentration of the kinase determined from this graph. Figure 3: Example of Kinase Titration at ATP K m,app 0.50 Kinase Titration at ATP K m,app 0.40 FRET ratio 0.30 0.20 0.10 0.00 0.001 0.01 0.1 1 10 100 1000 [NTRK1] ng/ml The EC 80 value determined from the example data was 8.7 ng/ml kinase. Based on this result, 9 ng/ml kinase was used to determine inhibitor IC 50 values when performing the assay at 223 µm ATP. 8

Step 4: Determination of Inhibitor IC 50 Value. (4.1) The general procedure for determining an inhibitor IC 50 value is as follows: 1. Add 2.5 µl of inhibitor in 4% DMSO at 4-fold the final assay concentration to triplicate assay wells. 2. To start the reaction, add 2.5 µl of kinase at 4-fold the final assay concentration, followed by 5 µl of substrate + ATP at 2-fold the final reaction concentrations. 3. The remainder of the protocol is similar to previous steps. (4.2) A dilution series of inhibitor in 100% DMSO is first prepared at 100 times the concentrations to be assayed. By performing the initial dilutions in 100% DMSO, solubility problems associated with dilutions into aqueous buffer can be minimized. This master dilution series of inhibitor can be prepared in two separate 8-tube PCR strips, and stored at - 20 or -80 for use in future experiments. The dilutions are staggered between strips as shown in the left side of Figure 4: 1. Add 50 µl of DMSO to tubes 2 8 of strip A, and all tubes of strip B. 2. Add 100 µl of inhibitor in DMSO, at 100-fold the highest concentration to be tested in the experiment, to tube 1 of strip A. 3. Transfer 50 µl of inhibitor from tube 1 of strip A to tube 1 of strip B. 4. After mixing, transfer 50 µl from tube 1 of strip B to tube 2 of strip A. 5. This process is repeated for all but the final tube of strip B, which contains only DMSO (no inhibitor). Figure 4: Preparing a Dilution Series of Inhibitor Strip A S trip B A B C D E F G H Column 1 Column 2 A B C D E F G H Column 1 123 A B C D E F G H Column 2 123 Master Dilutions 100x, 100% DMS O (PCR-Tube Strips) Intermediate Dilutions 4x, 4% DMS O (96-Well Plate) Assay Plate (384-Well Plate) Assay Plate (384-Well Plate) (4.3) From the master dilutions of inhibitor in 100% DMSO, intermediate dilutions are then prepared in two columns of a 96-well plate. The 96-well plate is used only as a convenient vessel for preparing the intermediate dilutions. 1. First, place 96 µl of kinase reaction buffer into all wells of two columns of a 96-well plate. 2. Then, transfer 4 µl of the master inhibitor stock from strip A into column 1 of the 96 well plate, and 4 µl of the master inhibitor stock from strip B into column 2 of the 96-well plate. 9

3. Mix the solutions well, either with a plate shaker or by mixing with a 20 µl multichannel pipette. 4. Using an 8-channel pipette, add 2.5 µl of inhibitor from the intermediate dilution in the 96-well plate to the 384-well assay plate as shown in figure 4. Use column 1 of the intermediate stock to fill rows A, C, E, etc. of the 384-well assay plate, and column 2 to fill the alternating rows B, D, F, etc. (4.4) Using the kinase dilution prepared in Step 3.1 as your stock, prepare a 1 ml solution of kinase in kinase reaction buffer at 4x the final desired reaction concentration of the kinase. From Step 3, 9 ng/ml kinase was determined to be the concentration required for the assay. Calculation: Kinase: Initial conc. = 200 ng/ml* 1x = 9 ng/ml 4x = 36 ng/ml * prepared in step 3.1 Add 180 µl of 200 ng/ml kinase to 820 µl kinase reaction buffer. (4.5) Add 2.5 µl of the kinase solution prepared in step 4.4 to each well of the assay plate. (4.6) In an appropriate container, prepare 1 ml of a solution of substrate and ATP in kinase reaction buffer at 2 times the final concentration of each reagent desired in the assay. If a 1000 µl solution is prepared in a plastic reagent reservoir (trough), then the next addition step can be performed using a multichannel pipette. Substrate: Stock = 30 µm 1x = 0.2 µm 2x = 0.4 µm ATP: Stock = 10 mm 1x = 223 µm 2x = 446 µm [Initial] [Final 4x] Kinase: 180 µl * 200 ng/ml = 1000 µl * 36 ng/ml 820 µl kinase reaction buffer Substrate: 13.3 µl * 30 µm = 1000 µl * 0.4 µm ATP: 44.6 µl * 10 mm = 1000 µl * 446 µm 942.1 µl kinase reaction buffer Add 13.3 µl of 30 µm substrate and 44.6 µl of 10 mm ATP to 942.1 µl kinase reaction buffer. (4.7) Start the kinase reaction by adding 5 µl of the substrate + ATP solution prepared in step 4.6 to each well of the assay plate and mix briefly, either by pipette or on a plate shaker. (4.8) Cover the assay plate and allow reaction to proceed for 1 hour at room temperature. (4.9) Prior to completion of the assay, prepare 1 ml of a solution of EDTA and Tb-labeled antibody at 2 times the desired final concentrations of each reagent in TR-FRET dilution buffer. The antibody is stable in EDTA for several hours, but because it is sensitive to high concentrations of EDTA, we recommend first adding the concentrated EDTA to the dilution buffer, mixing the solution well, and then adding the antibody before mixing further. 10